Despite many advances in the understanding of the molecular pathogenesis of human immunodeficiency virus type-1 (HIV-1), effective treatment of HIV-1 infection is still not available. Ribozymes (ribonucleotide enzymes) are RNA molecules that possess sequence specific endoribonuclease activity, and have potential utility as tools to study the molecular pathogenesis of HIV-1 and as anti-HIV-1 therapeutic agents. Although ribozyme structure-function relationships in vitro have been the subject of numerous investigations, before ribozymes can be applied as anti-HIV-1 therapies, a better understanding of the characteristic of ribozomes that confer efficient cleavage of target RNA in vivo is needed. The studies outlined in this proposal are designed to develop an easily manipulable system to study ribozyme structure-function relationship in vivo. This experimental system employs Moloney murine leukemia virus (MMLV) to intracellularly co-localized ribozyme and substrates. Assessment of ribozyme activity in vivo is based upon inhibition of expression of a lacZ reporter gene. This system will be used to study the effect of variations in the amount and symmetry of complementary base pairing between ribozyme and substrates on the activity of an anti-HIV-1 hammerhead ribozyme in vivo, and to determine which potential ribozyme cleavage sites in a HIV-1 gene transcript are the best ribozyme targets sites in vivo. The accessibility of ribozyme target sites as determined by this approach will be compared to results obtained by an RNase protection assay as outlined in core 1 of this application, and the anti- HIV-1 effect of biologically active ribozymes identified by the co- localization approach will be confirmed by assessment of their ability to inhibit HIV-1 replication. Lastly, anti-HIV-1 ribozymes employed in existing studies have been designed by a lengthy, systematic approach. The studies outlined in the later sections of this proposal will develop a method to select efficient anti-HIV-1 ribozymes from a pool of hammerhead ribozymes with randomly generated base-pairing flanking arms, based on their ability to interfere with HIV-1 cytopathicity in the tissue culture cell line. Although the studies outlined herein are designed to develop ribozymes as anti-HIV-1 therapeutics, the information obtained on the determinants of ribozyme activity in vivo are expected to contribute in more general way to the development of ribozyme technology.